Formalin has been used by the histology field for over half a century. When used at room temperature, formalin diffuses into a tissue section and cross-links proteins and nucleic acids, thereby halting metabolism, preserving biomolecules and readying the tissue for paraffin wax infiltration. In practice, formalin fixation primarily occurs at room temperature or higher. Some groups perform fixation at slightly elevated temperatures, presumably to increase the cross-linking rate. Just as heat increases cross-linking rate, cold formalin significantly decreases cross-linking rate. For this reason, histologists typically perform tissue fixation at room temperature or higher. Some groups have used cold formaldehyde, but only in specialized situations and not for fixing tissues. For instance, groups use cold formalin to examine lipid droplets or other special situations.
Several effects are observed in tissues that are either under exposed or over exposed to formalin. If a tissue sample is not treated with formalin for a sufficiently long period of time, tissue morphology is typically very poor when the tissues are subjected to standard tissue processing. For example, in inadequately fixed tissue, subsequent exposure to ethanol shrinks the cellular structures and condenses nuclei since the tissues will not have the chance to form a proper cross-linked lattice. When under fixed tissue is stained, such as with hematoxylin and eosin (H&E), many white spaces are observed in between the cells and tissue structures, condensed nuclei and loss of cytoplasm, and samples appear pink and unbalanced with the hematoxylin stain. Tissues that have been exposed to formalin too long typically do not work well for subsequent immunohistochemical processes, presumably because of nucleic acid and/or protein denaturation and degradation. As a result, the optimal antigen retrieval conditions for these tissues do not work properly and therefore the tissue samples appear to be under stained.
Proper medical diagnosis and patient safety require properly fixing the tissue samples prior to staining. Accordingly, guidelines have been established by oncologists and pathologists for proper fixation of tissue samples. For example, according to the American Society of Clinical Oncology (ASCO), the current guideline for fixation time in neutral buffered formalin solution for HER2 immunohistochemistry analysis is at least 6 hours, preferably more, and up to 72 hours. It would be advantageous to develop a process for rapidly fixing tissue samples both to better preserve biological molecules and tissue morphology before significant degradation occurs, and to provide accurate test results to medical professionals and patients as quickly as possible.
Such a process is particularly important for preserving post-translational modification signals. Post-translational modification of proteins plays an extremely important role in cellular metabolism. For instance, phosphorylation of proteins regulates many cellular functions such as cell cycle control, replication, transcription and translation. Other modifications like ubiquitination may target those proteins for degradation and have profound effects on cellular functions. Unfortunately, when the cell loses control of some of these modifications, cellular proliferation results and cancers arise. In fact, most cancer pathways are now being linked integrally with phosphorylation cascades that ultimately cause cells to become immortalized and diagnosed as cancerous. It will be extremely important for researchers and companies to understand if certain modifications exist in cancers as a way of diagnosing a particular cancer type and/or predicting treatment outcome.
Unfortunately, currently existing methods, typically requiring long fixation and processing times at room temperature, are not good for preserving protein modifications. The issue of losing modification signals of proteins has been addressed by many researchers in the past, for example, halting the action of phosphatases for example, Millipore (Kinase Inhibitor Cocktail) Cat #20-116), Thermo Scientific (Halt Phosphatase Inhibitor Cocktail) Cat#78420) etc.
Others in the industry have developed fast freezing methods in order to halt the action of modification enzymes (Lawson et. al. Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions, 2011, vol. 62, issue 2, pages 115-122). Fast freezing methods generally apply to the instances where whole organs need to be preserved for implantation and involve DMSO or sugars. Unfortunately, fast freezing may initially slow down the action of such enzymes but does not inhibit their action upon thawing of the sample. These approaches all have various limitations.
The above approaches are mainly useful for inhibiting modification enzymes in tissue extracts or cell lines due to the poor diffusion properties or lack of effectiveness of most phosphatase inhibitors in solid tissues. Larger molecular inhibitors such as proteins will have extremely low diffusion rates. Smaller molecules such as orthovanadate have higher diffusion rates but limited specificity and are not extremely effective against all phosphatases. There is not currently available a universal, easy to implement method of halting the action of modification enzymes effectively in solid tissue samples. Thus, it is desirable in the art to develop novel tissue fixing methods offering excellent quality in preserving tissue morphology, protein structure and/or post-translation modification signals.